血小板源性生长因子和血小板源性内皮细胞生长因子在内皮细胞和血管平滑肌细胞中的作用研究

摘要

目的：通过体外转染血小板源性生长因子（ PDGF）和血小板源性内皮细胞生长因子（ PD-ECGF）质粒，观察两种生长因子对人脐静脉内皮细胞EAHY926和主动脉血管平滑肌细胞T/G HA-VSMC的影响，评估两种生长因子对血管损伤治疗的可行性。方法构建人pcDNA 3.1（＋）空载（空载组）、pcDNA 3.1（＋）-PDGF-透明质酸（HA）（PPH 组）和 pcDNA 3.1（＋）- PD-ECGF - HA（PPEH 组）质粒，分别转染入 EAHY926和 T/G HA-VSMC中，四甲基偶氮唑盐微量酶反应比色法（ MTT法）检测内源性PD-ECGF和外源性PD-ECGF对EAHY926和T/G HA-VSMC细胞增殖的影响；细胞伤痕实验观察内源性PD-ECGF和外源性PD-ECGF对 EAHY926和T/G HA-VSMC细胞迁移速度的影响。结果 MTT 法检测3组 EAHY926吸光度值比较，差异有统计学意义（ F ＝235.18，P＜0.001），其中PPH组高于空载组和PPEH组（P＜0.05）；空载组与PPEH组比较，差异无统计学意义（P＞0.05）。MTT法检测3组T/G HA-VSMC吸光度值比较，差异有统计学意义（ F＝82.89，P＜0.001），其中PPH组高于空载组（P＜0.05）；PPEH组低于空载组（P＜0.05）。MTT法检测转染PPEH质粒的培养基（L-PPEH）对PPH诱导的EAHY926和T/G HA-VSMC增殖的影响，空载组、PPH组、PPH＋L-PPEH组EAHY926吸光度值比较，差异无统计学意义（F＝512.89，P＝0.183）。空载组、PPH组、PPH＋L-PPEH组T/G HA-VSMC吸光度值比较，差异有统计学意义（F＝317.40，P＜0.001）；其中PPH组高于空载组和PPH＋L-PPEH组（P＜0.05）。细胞伤痕实验证实，转染PPEH质粒的EAHY926、T/G HA-VSMC细胞迁移能力增强；PPEH组EAHY926平均细胞迁移距离百分比（56.1%±2.2%）较空载组（27.8%±3.4%）升高（t＝-15.08，P＜0.001）；PPEH组T/G HA-VSMC平均细胞迁移距离百分比（69.1%±2.3%）较空载组（43.6%±5.8%）升高（t＝-33.64，P＜0.001）。用转染PPEH质粒的培养基培养EAHY926、T/G HA-VSMC发现，外源性PD-ECGF处理的EAHY926、T/G HA-VSMC细胞迁移能力亦明显增强。结论通过转染PDGF质粒，模拟血管损伤后内皮细胞和平滑肌细胞受到PDGF和/或PD-ECGF刺激，发现 PDGF能明显上调内皮细胞和血管平滑肌的增殖和迁移。PD-ECGF对血管内皮细胞增殖的效果不明显，但能上调血管内皮细胞和血管平滑肌的迁移速度；同时PD-ECGF能抑制血管内皮细胞的增殖。两种因子同时使用能抑制平滑肌细胞的过度增殖，促进内皮细胞和平滑肌细胞的迁移。两者联合使用可能成为促进经皮冠状动脉介入术（ PCI）后损伤血管恢复及预防血管再狭窄的新策略。%Objective To investigate the influence of platelet derived growth factor( PDGF)and platelet -derived endothelial cell growth factor( PD-ECGF)on human umbilical endothelial cells EAHY926 and human-aorta vascular smooth muscle cells T/G HA-VSMC by transfecting the plasmids of PDGF and PD-ECGF in vitro and to evaluate the possibility of treating vascular injury by the two kinds of plasmids. Methods pcDNA 3. 1( +)no-load(no-load group),pcDNA 3. 1 ( +) -PDGF-HA( PPH group)and pcDNA 3. 1( +) -PD-ECGF-HA( PPEH group)plasmids were constructed and transfected into EAHY926 and T/G HA-VSMC. MTT method was used to evaluate the influence of endogenous and exogenous PD-ECGF on the proliferation of EAHY926 and T/G HA-VSMC before and after transfection. Cell wound healing assay was used to evaluate the influence of endogenous and exogenous PD-ECGF on the migration speed of EAHY926 and T/G HA-VSMC after transfection. Results MTT method was used to detect the absorbance value of EAHY926 among the three groups,and the differences were statistically significant(F=235. 18,P<0. 001),with the PPH group significantly higher than the no-load group and PPEH group( P <0. 05 ) . The no - load group and PPEH group showed no statistically significant differences(P>0. 05). MTT method was also used to detect the absorbance value of T/G HA-VSMC among the three groups, and the differences were statistically significant(F=82. 89,P<0. 001),with the PPH group significantly higher than the no-load group(P<0. 05),and the PPEH group significantly lower than the no-load group(P<0. 05). MTT method detecting the influence of medium transfected with PPEH on the proliferation of EAHY926 and T/G HA-VSMC induced by PPH. There was no significant difference in the absorbance value of EAHY926 among no-load group,PPH group and PPH+L-PPEH group ( F=512. 89,P=0. 183). There was significant difference in the absorbance value of T/G HA-VSMC among no-load group, PPH group and PPH+L-PPEH group(F=317. 40,P<0. 001),with the PPH group significantly higher than the no-load group and PPH+L-PPEH group ( P <0. 05 ) . Cell wound healing assay demonstrated that EAHY926 and T/G HA-VSMC transfected with PPEH plasmid had a higher migration ability;compared with the no-load group,the average percentage of cell migration distance of EAHY926 in PPEH group was significantly increased〔(56. 1% ±2. 2%)vs. (27. 8% ±3. 4%)〕(t=-15. 08,P < 0. 001 );compared with the no - load group, the average percentage of cell migration distance of T/G HA-VSMC in PPEH group was significantly increased〔(69. 1% ±2. 3%)vs. (43. 6% ±5. 8%)〕(t= -33. 64,P<0. 001). EAHY926 and T/G HA-VSMC cultured in medium transfected with PPEH showed that the migration ability of EAHY926 and T/G HA-VSMC transfected with PPEH processed by exogenous PD-ECGF also increased significantly. Conclusion After transfection of PDGF plasmid,the stimulation of PDGF and/or PD-ECGF on endothelial cells and smooth muscle cells is simulated. It is found that PDGF can significantly up - regulate the proliferation and migration of endothelial cells and smooth muscle cells. PD-ECGF has limited influence on the proliferation of vascular endothelial cells,but can up-regulate the migration speed of vascular endothelial cells and vascular smooth muscle cells. Meanwhile,PD-ECGF can inhibit the proliferation of vascular endothelial cells. The combined application of the two factors can inhibit over-proliferation of smooth muscle cells and promote migration of endothelial cells and smooth muscle cells. The combination may become a new strategy for the recovery of vascular damage and prevention of vascular restenosis after PCI.